70-kda Heat Shock Proteins And Their Associated Cofactors

我们的实验室对细胞中正常和病理蛋白质复合体的形成和溶解感兴趣，并强调分子伴侣在这一过程中的作用。我们继续我们的研究，通过建立HSC70辅伴侣、神经元特异性生长素和普遍表达的GAK的敲除小鼠来研究Hsc70在笼蛋白介导的内吞作用中的作用。这些分子伴侣将分子筛呈现给Hsc70，因此在从包被分子筛蛋白的囊泡中剥离分子筛蛋白和在胞浆中分子伴侣分子筛子蛋白方面起着重要作用。为了敲除GAK，我们必须制造一只有条件的敲除小鼠，因为传统的敲除是胚胎致死的。我们的GAK基因敲除研究表明，GAK在发育过程中和成年小鼠中是一个必不可少的基因。至于神经元特异性的生长素，新生的基因敲除小鼠的死亡率相对较高，存活的小鼠通常比野生型小鼠的体重更低。存活的小鼠有正常的寿命，但它们之所以存活下来，可能是因为存活的新生和成年生长素基因敲除小鼠的大脑中GAK上调了3倍。令人惊讶的是，正常水平的GAK不能弥补缺乏生长素，正如观察到的那样，笼蛋白介导的内吞作用在基因敲除小鼠的海马神经元中受到抑制。无论是在原代培养的神经元还是在原位培养的突触中，敲除突触上都出现了更多的笼状蛋白包裹的囊泡和空笼。这些结果表明，生长素在回收大脑中的突触小泡方面具有特殊的作用，这只能通过显著上调小鼠神经元中的GAK水平来弥补。 从该条件性GAK基因敲除小鼠中分离出小鼠胚胎成纤维细胞(MEF)，并通过表达Cre重组酶的腺病毒对MEF中的GAK进行干扰。敲除GAK后，可完全阻断MEF中络合蛋白介导的内吞作用。此外，MEF的网状蛋白结构被完全破坏，从质膜和跨高尔基网络中都失去了网状蛋白。为了弥补胞苷介导的内吞作用的损失，液体相摄取显著增加。有趣的是，在没有GAK的情况下，细胞分裂被阻止，但MEF仍然存活了几周。这些结果表明，GAK的缺失以及Hsc70在胞浆中缺乏对细胞内多个系统的分子伴侣作用。 除了我们对网状蛋白介导的内吞作用的研究外，我们还研究了在酵母和哺乳动物细胞中可以错误折叠为淀粉样构象的感染性蛋白质--普恩的繁殖。在酵母中，分子伴侣Hsp104调节包括PSI+在内的几种酵母蛋白的遗传，PSI+是翻译终止因子Sup35p的蛋白形式。通过使用活细胞成像，我们发现HSP104不仅切断了Prion种子，而且还修剪或缩小了种子的大小。我们发现，当HSP104通过在胍中培养酵母来灭活时，种子变得更小，因为HSP104仍然能够修剪它们。然而，它不能切断它们，种子数量减少，并发生了Pron固化。然而，当Hsp104通过表达显性负的Hsp104突变体而完全失活时，Hsp104不再修剪或切断种子，因此随着腌制的进行，种子的大小增加，数量减少。因此，HSP104既可以修剪种子以保持其稳定的大小，也可以在酵母分裂时切断种子以保持稳定的种子数量。

Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in this process. We have continued our studies on the role of Hsc70 in clathrin-mediated endocytosis by generating knockout mice of the HSC70 cochaperones, neuronal-specific auxilin and ubiquitously expressed GAK. These chaperones present clathrin to Hsc70 and therefore have a major role in uncoating clathrin from clathrin coated vesicles and chaperoning clathrin in the cytosol. To knock-out GAK, we had to make a conditional knock-out mouse since the conventional knock-out was embryonic lethal. Our GAK knockout studies showed that GAK was an essential gene during development and in adult mice. As for neuronal-specific auxilin, the newborn knockout mice had relatively high mortality and surviving pups generally had a lower body weight than the wild-type pups. The surviving mice had a normal life-span, but they probably survived only because GAK was up-regulated as much as 3-fold in the brains of both surviving neonate and adult auxilin knockout mice. Surprisingly, normal levels of GAK could not compensate for the absence of auxilin as shown by the observation that clathrin-mediated endocytosis was inhibited in the hippocampal neurons of the knockout mice. An increased number of clathrin-coated vesicles and empty cages were present at the knockout synapses both in situ and in primary neuronal cultures. These results show that auxilin has a specialized role in recycling synaptic vesicles in the brain, which can only be compensated for by marked up-regulation of GAK levels in mouse neurons Mouse embryonic fibroblasts (MEFs) were derived from this conditional GAK knockout mouse and GAK was disrupted in the MEFs by using adenovirus expressing Cre recombinase. Knocking-out GAK, completely blocked clathrin-mediated endocytosis in the MEFs. In addition, the clathrin organization of the MEFs was totally disrupted with loss of clathrin from both the plasma membrane and trans-Golgi network. To compensate for the loss of clathrin-mediated endocytosis, there was a marked increase in fluid phase uptake. Interestingly, in the absence of GAK, cell division was arrested, but the MEFs were still viable for several weeks. These results show that the absence of GAK and in turn the lack of chaperoning of clathrin in the cytosol by Hsc70 had a profound effect on multiple systems in the cell. In addition to our research on clathrin-mediated endocytosis, we also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in yeast and mammalian cells. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. By using live cell imaging, we found that Hsp104 not only severs prion seeds, but trims or reduces the size of the seeds. We found that when Hsp104 was inactivated by growing yeast in guanidine, the seeds became smaller because Hsp104 was still able to trim them. However, it could not sever them and the number of seeds decreases and prion curing occurred. However, when Hsp104 was completely inactivated by expressing a dominant negative Hsp104 mutant, Hsp104 no longer trimmed or severed the seeds and therefore the seeds both increased in size and decreased in number as curing occurred. Therefore, Hsp104 both trims the seed to maintain their steady-state size and severs them to maintain the steady-state number of seeds as the yeast divides.